85 ADDITION OF REDUCED GLUTATHIONE TO FREEZING MEDIUM IMPROVED THE SPERM FUNCTIONALITY OF THAWED BOAR SPERMATOZOA
J. Gadea A , F. Garcia-Vázquez A , D. Gumbao A , F.A. Rodríguez A and C. Matás AADepartment of Physiology, University of Murcia, Murcia, Spain. Email: jgadea@um.es
Reproduction, Fertility and Development 17(2) 192-193 https://doi.org/10.1071/RDv17n2Ab85
Submitted: 1 August 2004 Accepted: 1 October 2004 Published: 1 January 2005
Abstract
The processes of cooling and freezing/thawing produce physical and chemical stresses on the sperm membrane that are associated with the oxidative stress and reactive oxygen species (ROS) generation that reduce sperm viability and fertilizing ability. It is known that the process of freezing is associated with a significant reduction of the GSH content in boar sperm (Gadea et al. 2004 Theriogenology 62, 690–701). The addition of antioxidants to freezing medium could prevent the formation of ROS and improve the seminal parameters. The aim of these experiments was to investigate the effects of the addition of reduced glutathione (GSH) to freezing extenders on capacitation status and changes in the sulfhydryl groups of proteins on the sperm surface. Ejaculate-rich fractions from three mature boars were frozen by classic methodology (Westendorf et al. 1975 Dtsch. Tierarztl Wochenschr. 82, 393–395) using lactose/egg-yolk extender with 0 mM (group 0), 1 mM (group 1), or 5 mM (group 5) GSH. To detect increase in plasma membrane lipid packing disorder, sperm samples were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Reprod. Dev. 45, 378–391). Cells were classified as low M540 (viable, noncapacitated), high M540 (viable, capacitated), or Yo-Pro-1 positive (dead sperm) using flow cytometry. The sulfhydryl status of proteins from spermatozoa surface was evaluated with a fluorescent stain 5-iodoacetamidofluoresceine (5-IAF). The addition of GSH to the freezing medium had a positive influence on the parameters studied, increasing the proportion of viable noncapacitated spermatozoa and reducing the number of dead with a similar number of viable capacitated (Table 1). The proportion of spermatozoa stained by 5-IAF was significantly lower when GSH was added. In conclusion, we can assume that the addition of reduced glutathione to the freezing medium had a protective effect on spermatozoa functionality.
|
This work was supported by AGL- 2003-03144.
